Vorremmo ringraziare Philippa Warren per il suo gentile aiuto.

1. Contorno di analisi: <ol><li> Preparazione di base: le diapositive Camera sono rivestiti con poli-D-lisina vetrini durante la notte e sterile vengono preparati prima di iniziare l&#39;esperimento. Terreno è preparato utilizzando Media Dulbecco Modified Eagle (DMEM), integrato con il 10% di siero fetale bovino (FCS), e 1% di penicillina-streptomicina-Fungizon (FPF). Anche una bottiglia di calcio / magnesio soluzione salina bilanciata libero Hanks (HBSS) è preparato. </li><li> Primarie di cellule di Schwann ratto coltivate in matraccio trypsinized per 3 minuti con 0,1% tripsina. Primaria cultura astrociti di ratto è trypsinized per 5 minuti con 0,1% tripsina. Tripsina è inactived aggiungendo DMEM supplementato con 10% FCS e 1% FPF. </li><li> Trypsinized cellule di Schwann e gli astrociti vengono trasferiti a separare i tubi falcon da 15 ml e centrifugato a 300G per 5 minuti. </li><li> Cellule di Schwann e gli astrociti sono risospese alla densità di 2 x 10 6 (vedere la sezione dei risultati rappresentativi delle variazioni di densità delle cellule). </li><li> 50 goccia l di sospensione delle cellule di Schwann è posta ad una estremità di pozzi camera PDL rivestiti di diapositive. Una striscia di vetro è stato utilizzato per spalmare la caduta verso il centro della camera per generare un bordo dritto. (Utilizzando un tagliatore di vetro, un coprioggetto di vetro rettangolare vengono tagliate longitudinalmente in modo che fosse in base alla larghezza della camera di diapositive utilizzate per le analisi di confine) </li><li> Una seconda goccia di 50 ml di sospensione astrociti è stato posto dalla parte opposta del pozzo stesso e spalmato verso il centro per creare un bordo dritto parallelo alla prima goccia striscio con un piccolo spacco 0,2 millimetri tra di loro. </li><li> La camera di diapositive contenenti le gocce sono posti in incubatore a 37 ° C per 2 ore. </li><li> Dopo 2 ore, le culture sono stati lavati con DMEM per rimuovere non iscritti detriti cellulari e mezzo fresco contenente DMEM / 10% FCS / 1% PSF aggiunto. Nota: DMEM / 10% FCS / 1% PSF può essere completato con forskolina (2μM) ed estratto di ipofisi bovina (BPE) (10μg / mL) a stimolare la proliferazione delle cellule di Schwann. BPE fornirà i fattori di crescita necessari per consentire a lungo termine cultura delle cellule di Schwann e forskolina aumenta i livelli di adenosina monofosfato ciclico all&#39;interno delle cellule di Schwann di indurre la proliferazione </li><li> Le cellule sono coltivate per circa 8-10 giorni a 37 ° C, 7% di CO 2 incubatore. Il mezzo deve essere cambiata ogni 3 giorni. I due fronti cellula alla fine raggiungere l&#39;altro e un confine netto si forma tra i due tipi di cellule. In questa fase gli esperimenti possono essere effettuate analisi di fattori coinvolti nella formazione di confine. </li><li> Co-coltura può essere fissato con 4% di formaldeide e le cellule di Schwann può essere immunoistochimica con anticorpi anti-P75 e astrociti con anticorpi anti-GFAP per identificare le cellule in co-coltura. P75 anticorpo riconosce a bassa affinità dei recettori NGF su cellule di Schwann che gli astrociti mancanza. Anti-GFAP anticorpo riconosce proteina acida gliale firbrillary che localizza al citoplasma astrociti. (Si veda la Figura 1A per contorno rappresentante) </li></ol> 2. Migrazione di analisi: <ol><li> 4 piastre bene (15mm pozzi) sono coperti per una notte PDL e preparato per la creazione di monostrati astrociti. </li><li> Primaria cultura astrociti è trypsinized per 5 minuti con 0,1% tripsina. Tripsina viene inattivato con l&#39;aggiunta di DMEM supplementato con 10% FCS e 1% PSF e cellulari centrifugati a 300G per 5 minuti. (Vedi figura 2) </li><li> Astrociti vengono nuovamente sospesi a 1x10 5 cellule / ml. 1 ml di questa soluzione è aggiunto ad ogni rivestito PDL bene in 4 piatti bene. Astrociti sono in coltura per 24-48 ore fino a quando il monostrato è completamente confluenti. </li><li> Per valutare la migrazione delle cellule di Schwann su monostrati astrociti, parallelamente alla creazione di monostrati astrociti, circolare PDL rivestite coprioggetto di vetro sono posti in una provetta da 50 ml e schiacciato con una pipetta di plastica per creare piccoli frammenti di vetro. </li><li> Frammenti di vetro vengono trasferiti in un pozzo in 6 piatto piatto bene e usando pinze adatte frammenti imprese sono scelti e collocati in altri pozzi. 5 frammenti di vetro vengono inseriti in ciascun pozzetto in 6 piastre. </li><li> Primarie di cellule di Schwann coltivate in matraccio trypsinized per 3 minuti con 0,1% tripsina. Trypsinized cellule di Schwann sono trasferiti a separare tubo falcon da 15 ml e centrifugato a 300 g per 5 minuti. Cellule di Schwann sono risospese a 2x 10 6 cellule / ml in DMEM/10% FCS / 1% FPF. </li><li> 20 gocce l di sospensione delle cellule di Schwann si aggiunge oltre ogni 5 frammenti coprioggetto posti in 6 piastre e le cellule vengono incubate a 37 ° C CO, 7% 2 per 2 ore. </li><li> Dopo 2 ore, le cellule di Schwann sono collegati ai frammenti coprioggetto e DMEM/10% FCS / 1% PSF (Integrato con 2μM di forskolina e 10μg / mL di BPE è aggiunto a ciascun bene in modo che i frammenti sono completamente coperti di media) . </li><li> I 6 piastre contenenti i frammenti vengono poi messi in incubatrice per 24-48 ore fino a quando i frammenti di vetro sono completamente confluente con cellule di Schwann. </li><li> Una volta che i frammenti sono confluenti con le cellule di Schwann, ogni frammento viene raccolto con cura utilizzando pinze taglienti e immersa in HBSS per rimuovere le cellule distaccati e poi faccia rovesciata verso il basso a monostrato astrociti. </li><li> Ogni bene è poi coperto con 1 ml di DMEM/10% sono consentiti FCS / 1% di cellule di Schwann PSF e di migrare dalle 24 alle 48 periodo. Durante questo periodo reagenti sperimentali come fattori di crescita possono essere aggiunti per valutare il loro effetto sul numero e la distanza di migrazione delle cellule di Schwann. </li><li> Dopo il periodo di migrazione, i frammenti di vetro sono fissati al fondo di ciascun pozzetto con l&#39;aggiunta del 4% paraformaldeide (PFA) per 30 minuti. A seguito di fissazione, immunostain le cellule di Schwann di p75 (riconoscimento del recettore a bassa affinità NGF) poi li macchia con ABC strepavidin kit-diaminobenzidina (DAB), per consentire la visualizzazione delle cellule di Schwann sotto microscopio ottico. </li></ol> 3. Rappresentante dei risultati: A seguito di 8-10 giorni di Schwann-co-culture astrociti mostrano un confine netto tra i due tipi di cellule. Questo confine è più marcato se forskolina e BPE è usato nella cultura, come la proliferazione delle cellule di Schwann è migliorata. Se nessun confine è osservata durante il periodo di 10 giorni, il tempo della cultura può essere aumentata ulteriormente fino a quando i confini sono stabiliti. Modifica del rapporto di densità delle cellule è un modo alternativo per aumentare la formazione di confine. Un rapporto di 3:1 di Schwann: la densità delle cellule astrociti possono essere utilizzati per la preparazione sospensioni cellulari 8,18. A seguito di immunostaining per identificare le cellule di Schwann e gli astrociti, cellule mescolanza di valutare una linea possono essere tratte dove il confine è stabilito ed il numero di cellule di Schwann migrazione nel territorio astrociti possono essere contate al microscopio a fluorescenza. Di seguito sono riportati due esempi di immagini ottenute confini, uno con ben formato segregazione delle cellule di Schwann e gli astrociti (Figura 1A) e uno in cui le cellule non hanno segregato e in diversi territori a causa della scarsa tecnica (Figura 1B). <img src="/files/ftp_upload/2214/2214fig2.jpg" alt="Figura 1" /> . Figura 1 test Boundary: Schwann-astrociti interazione. A) Un giorno 10 co-coltura saggio in presenza di forskolin e BPE per stimolare la proliferazione delle cellule di Schwann dimostra un confine netto tra i due tipi di cellule (rosso = P75, verde = GFAP). B) Un giorno 10 co-coltura saggio in cui il confine non è formato e le due fronti cella mista. Ciò era dovuto alla miscelazione dei due gocce pur ponendo gocce astrociti e cellule di Schwann uno accanto all&#39;altro durante la preparazione del confine (rosso = P75, verde = GFAP). <img src="/files/ftp_upload/2214/2214fig1.jpg" alt="Figura 2" /> Figura 2. Un&#39;immagine rappresentativa della confluenti monostrato astrociti Saggi di migrazione valutare il movimento di un tipo di cellule sulla superficie di un altro tipo di cellula e quindi valutare un fenomeno diverso da quello della formazione di confine. La pratica è necessaria per garantire invertendo i coprioggetti su monostrati non porta a danni alla cellule di Schwann-coperto frammenti di vetro e monostrati astrociti dal pinze taglienti. Per valutare la migrazione delle cellule di Schwann, la distanza massima della migrazione e del numero di cellule migrano dal bordo del vetrino può essere misurato con microscopio ottico. Di seguito l&#39;immagine di cellule di Schwann colorate con anticorpi P75 con colorazione DAB sulla superficie del monostrato astrociti (Figura 3). <img src="/files/ftp_upload/2214/2214fig3.jpg" alt="Figura 3" /> Figura 3. Test di migrazione utilizzando test invertita coprioggetto. A) cellule di Schwann (marrone, immunoistochimica con anticorpi p75) migrare lontano dal bordo del vetrino. La freccia indica la direzione della migrazione delle cellule dal vetrino.

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cells+transplanted+into+normal+and+X-irradiated+adult+white+matter+do+not+migrate+extensively+and+show+poor+long-term+survival.">Schwann cells transplanted into normal and X-irradiated adult white matter do not migrate extensively and show poor long-term survival.</a> Exp Neurol. 164, 292-302 (2000).</li><li>Lakatos, A., Franklin, R. J., Barnett, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+ensheathing+cells+and+Schwann+cells+differ+in+their+in+vitro+interactions+with+astrocytes.">Olfactory ensheathing cells and Schwann cells differ in their in vitro interactions with astrocytes.</a> Glia. 32, 214-225 (2000).</li><li>Lakatos, A., Barnett, S. C., Franklin, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+ensheathing+cells+induce+less+host+astrocyte+response+and+chondroitin+sulphate+proteoglycan+expression+than+Schwann+cells+following+transplantation+into+adult+CNS+white+matter.">Olfactory ensheathing cells induce less host astrocyte response and chondroitin sulphate proteoglycan expression than Schwann cells following transplantation into adult CNS white matter.</a> Exp Neurol. 184, 237-246 (2003).</li><li>Ghirnikar, R. S., Eng, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-Schwann+cell+interactions+in+culture.">Astrocyte-Schwann cell interactions in culture.</a> Glia. 11, 367-377 (1994).</li><li>Ghirnikar, R. S., Eng, L. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrogliosis+limits+the+integration+of+peripheral+nerve+grafts+into+the+spinal+cord.">Astrogliosis limits the integration of peripheral nerve grafts into the spinal cord.</a> Brain Res. 277, 175-180 (1983).</li><li>Fok-Seang, J., Mathews, G. A., ffrench-Constant, C., Trotter, J., Fawcett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+of+oligodendrocyte+precursors+on+astrocytes+and+meningeal+cells.">Migration of oligodendrocyte precursors on astrocytes and meningeal cells.</a> Dev Biol. 171, 1-15 (1995).</li><li>Wilby, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Cadherin+inhibits+Schwann+cell+migration+on+astrocytes.">N-Cadherin inhibits Schwann cell migration on astrocytes.</a> Mol Cell Neurosci. 14, 66-84 (1999).</li><li>Grimpe, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+proteoglycans+in+Schwann+cell/astrocyte+interactions+and+in+regeneration+failure+at+PNS/CNS+interfaces.">The role of proteoglycans in Schwann cell/astrocyte interactions and in regeneration failure at PNS/CNS interfaces.</a> Mol Cell Neurosci. 28, 18-29 (2005).</li><li>Afshari, F. T., Kwok, J. C., White, L., Fawcett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+migration+is+integrin-dependent+and+inhibited+by+astrocyte-produced.">Schwann cell migration is integrin-dependent and inhibited by astrocyte-produced.</a> Glia. , (2010).</li><li>Santos-Silva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF/heparin+differentially+regulates+Schwann+cell+and+olfactory+ensheathing+cell+interactions+with+astrocytes:+a+role+in+astrocytosis.">FGF/heparin differentially regulates Schwann cell and olfactory ensheathing cell interactions with astrocytes: a role in astrocytosis.</a> J Neurosci. 27, 7154-7167 (2007).</li><li>Afshari, F. T., Kwok, J. C., Fawcett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-produced+ephrins+inhibit+schwann+cell+migration+via+VAV2+signaling.">Astrocyte-produced ephrins inhibit schwann cell migration via VAV2 signaling.</a> J Neurosci. 30, 4246-4255 (2007).</li></ol>

Nessun conflitto di interessi dichiarati.

I saggi di cui sopra sono stati utilizzati in diversi studi che dimostrano il ruolo dei molteplici fattori coinvolti nella formazione di confine tra il Schwann-astrociti e limitato la migrazione delle cellule di Schwann in ambiente astrocitaria. Comprendere la meccanismi alla base di queste manifestazioni è essenziale in quanto consentirebbe lo sviluppo di strategie per ottimizzare e migliorare l&#39;integrazione di innesti cell Schwann dopo il trapianto e in tal modo di facilitare l&#39;us...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41966-029</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14170-088</td>
<td>&nbsp;</td>
<tr>
<td>FCS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10091-148</td>
<td>&nbsp;</td>
<tr>
<td>PSF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15240-062</td>
<td>&nbsp;</td>
<tr>
<td>BPE</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13028-014</td>
<td>&nbsp;</td>
<tr>
<td>Forskolin</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>344273</td>
<td>&nbsp;</td>
<tr>
<td>Coverlips</td>
<td>&nbsp;</td>
<td>VWR-International</td>
<td>631-0149</td>
<td>&nbsp;</td>
<tr>
<td>Coverlips(rectangle)</td>
<td>&nbsp;</td>
<td>MENZEL-GLASER (Supplied by Thermo Scientifics)</td>
<td>BB022050A1</td>
<td>&nbsp;</td>
<tr>
<td>Chamber slides</td>
<td>&nbsp;</td>
<td>Nunc-LabTek </td>
<td>177380</td>
<td>&nbsp;</td>
<tr>
<td>4 well plates, 6 well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>&nbsp;</td>
<td>4 well plates 
6 well plates</td>
</tr>
<tr>
<td>rat p75 antibody</td>
<td>&nbsp;</td>
<td>Millipore, Temecula,California</td>
<td>MAB365</td>
<td>&nbsp;</td>
<tr>
<td>GFAP antibody</td>
<td>&nbsp;</td>
<td>Dako</td>
<td>Z0334</td>
<td>&nbsp;</td>
<tr>
<td>Secondary antibodies (Alexa-conjugated)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-11004 A-11034</td>
<td>Goat anti mouse 568 
Goat anti Rabbit 488</td>
</tr>
<tr>
<td>Secondary biotinylated </td>
<td>&nbsp;</td>
<td>Vector</td>
<td>&nbsp;</td>
<td>Goat anti mouse</td>
</tr>
<tr>
<td>DAB tablets </td>
<td>&nbsp;</td>
<td>Sigma-aldrich</td>
<td>D4418</td>
<td>&nbsp;</td>
<tr>
<td>ABC elite kit</td>
<td>&nbsp;</td>
<td>Vector</td>
<td>PK-6100</td>
<td>&nbsp;</td>
<tr>
<td>Fine Forceps </td>
<td>&nbsp;</td>
<td>FST</td>
<td>11295-10</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P6168</td>
<td>&nbsp;</td>
<tr>
<td>Fluorosave</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>345789</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

analysis of schwann-astrocyte interactions using in vitro assays

Cellule di Schwann sono una delle celle utilizzate nelle strategie di riparazione dopo le lesioni del midollo spinale. Cellule di Schwann sono in grado di supportare la rigenerazione assonale e germinazione da 1,2 secernono fattori di crescita e fornendo la promozione della crescita molecole di adesione 3 e molecole della matrice extracellulare 4. Inoltre essi mielinizzare gli assoni demielinizzati nel sito di lesione 5. Tuttavia dopo il trapianto, le cellule di Schwann non migrano dal sito di impianto e non si mescolano con il astrociti ospite 6,7. Ciò si traduce nella formazione di un confine netto tra le cellule di Schwann e gli astrociti, creando un ostacolo per gli assoni in crescita cercando di uscire dal retro innesto nel tessuto ospite prossimale e distale. Astrociti in contatto con le cellule di Schwann anche subire l&#39;ipertrofia e up-regolazione delle molecole inibitori 8-13. Test in vitro sono stati utilizzati per modellare cellule di Schwann-astrociti interazioni e sono stati importanti per capire il meccanismo alla base del comportamento cellulare. Questi test in vitro includono test di confine, dove viene effettuata una co-coltura con due cellule diverse con ogni tipo di cellula che occupa diversi territori con solo un piccolo spazio che separa i due fronti della cella. Come le cellule si dividono e migrano, i due fronti cellulare avvicinarsi gli uni agli altri e, infine, si scontrano. Questo consente il comportamento delle due popolazioni cellulari da analizzare al confine. Un&#39;altra variante della stessa tecnica è quello di mescolare le due popolazioni cellulari in coltura e nel corso del tempo i due tipi di cellule segregare con le cellule di Schwann insieme a blocchi come isole in mezzo astrociti insieme creazione di più di Schwann-astrociti confini. Secondo test utilizzato per studiare l&#39;interazione di due tipi di cellule è il test di migrazione in cui il movimento cellulari possono essere rintracciati sulla superficie del tipo di cellula monostrato 14,15. Questo test è comunemente noto come test coprioggetto invertita. Cellule di Schwann sono coltivati ​​su piccoli frammenti di vetro e sono invertiti a faccia in giù sulla superficie dei monostrati astrociti e la migrazione viene valutata dal bordo del vetrino. Entrambi i saggi sono stati strumentali per lo studio dei meccanismi connessi alla esclusione cellulare e la formazione di confine. Alcune delle molecole identificate utilizzando queste tecniche includono N-caderine 15, proteoglicani Condroitin Solfato (CSPGs) 16,17, FGF / eparina 18, Ef / efrine 19. Questo articolo si propone di descrivere test confine e il dosaggio della migrazione in modo graduale e chiarire gli eventuali problemi tecnici che potrebbero verificarsi.

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Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Questo articolo si propone di descrivere in modo graduale comunemente usato<em&gt; In vitro</em&gt; I test eseguiti nello studio delle cellule di Schwann-asrtocyte interazioni.

Analisi di Schwann-astrociti Interazioni utilizzo In Vitro Saggi

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

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Research

JoVE Journal

Biology

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

12.1K Views.  University of Cambridge. Questo articolo si propone di descrivere in modo graduale comunemente usato In vitro I test eseguiti nello studio delle cellule di Schwann-asrtocyte interazioni.

Video: Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

Rapida rilevazione omogenea dei saggi biologici Utilizzando Magnetic biopercezione sistema di modulazione

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Ricerca

Biologia

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

L&#39;analisi in vitro dei complessi CFTR PDZ-dipendenti macromolecolari di segnalazione

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Nella migrazione delle cellule in vitro e saggi Invasion

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3&#039; End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

Analisi delle reazioni RNA di trasformazione che utilizzano cellulari Sistemi gratis: 3 &#39;Fine Decolleté di pre-mRNA Substrati<em&gt; In vitro</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

In Vitro aggregazione Assays Utilizzando iperfosforilata Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Analisi comparativa di ormone umano della crescita nel siero Utilizzando SPRI, Nano-SPRI e saggi ELISA

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Protocollo di laboratorio per analisi genetiche del contenuto dell'intestino del rDNA acquatica macroinvertebrati Using Group-specific Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Effetto di proteine fluorescenti su Fusion partner utilizzando saggi di tossicità di Polyglutamine in lievito

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

Analisi microbiota utilizzando PCR in due fasi e sequenziamento genico rRNA 16S di nuova generazione

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In vitro Analisi della funzione E3 Ubiquitina ligasi

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...